Showing papers on "RAPD published in 2003"

Phylogenetic diversity and relationships among sorghum accessions using SSRs and RAPDs

Abstract: Two DNA-based fingerprinting techniques, simple sequence repeats (SSR) and random amplified polymorphic DNA (RAPD) analyses, were applied in sorghum germplasm analysis to compare suitability for quantifying genetic diversity. Twenty-two sorghum genotypes, representing an array of germplasm sources with important agronomic traits, were assayed for polymorphism using 32 RAPD primers and 28 sets of sorghum SSR primers. The results indicated that SSR markers were highly polymorphic with an average of 4.5 alleles per primer. The RAPD primers were less polymorphic with nearly 40% of the fragments being monomorphic. An analysis of genetic diversity among sorghum lines indicated that the genetic distances calculated from SSR data were highly correlated with the distances based on the geographic origin and race classifications. Based on the results of these studies, SSR markers appear to be particularly useful for the estimation of genetic similarity among diverse genotypes of sorghum. Key words : cluster, diversity, polymorphism, RAPD, Sorghum, SSR. African Journal of Biotechnology Vol.2(10) 2003: 334-340

Molecular Marker based Genetic Diversity Analysis in Rice ( Oryza sativa L.) using RAPD and SSR markers

Abstract: The availability of an array of molecular marker systems allowed comparing the efficiency of two of these marker systems to estimate the relationships among various taxa. The objective of this study was to assess the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. Out of 38 SSR primer pairs used, only one locus viz., RM115 was monomorphic. The average Polymorphism Information Content (PIC) value was 0.578 and it ranged from a low of zero (RM 115) to a high of 0.890 (RM 202). The Mantel matrix correspondence test was used to compare the similarity matrices and the correlation coefficient was 0. 582. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers.

Comparative assessment of DNA fingerprinting techniques (RAPD, ISSR and AFLP) for genetic analysis of cashew (Anacardium occidentale L.) accessions of India.

Abstract: Nineteen cashew accessions were analysed with 50 random primers, 12 ISSR primers and 6 AFLP primer pairs to compare the efficiency and utility of these techniques for detecting variation in cashew ...

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales.

Abstract: DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.

Inter simple sequence repeat (ISSR) markers as a tool for the assessment of both genetic diversity and gene pool origin in common bean (Phaseolus vulgaris L.)

Abstract: In this study, we report the use of ISSR to assess genetic diversity and to determine the relationships among ten cultivars of common bean developed in Argentina and three materials from France. ISSR markers resolved two major groups corresponding to the Andean and Mesoamerican gene pools of common bean. We compared the results of previous analysis, performed with RAPD markers (Galvan et al., 2001), with the results generated by means of ISSR. It appears that ISSR are better tools than RAPDs to identify beans by gene pool of origin though they did not revealed as many differences between individuals as RAPDs.

A first linkage map of olive (Olea europaea L.) cultivars using RAPD, AFLP, RFLP and SSR markers

Abstract: The first linkage map of the olive (Olea europaea L.) genome has been constructed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) as dominant markers and a few restriction fragment length polymorphisms (RFLP) and simple-sequence repeats (SSR) as codominant markers. Ninety-five individuals of a cross progeny derived from two highly heterozygous olive cultivars, Leccino and Dolce Agogia, were used by applying the pseudo test-cross strategy. From 61 RAPD primers 279 markers were obtained - 158 were scored for Leccino and 121 for Dolce Agogia. Twenty-one AFLP primer combinations gave 304 useful markers - 160 heterozygous in Leccino and 144 heterozygous in Dolce Agogia. In the Leccino map 249 markers (110 RAPD, 127 AFLP, 8 RFLP and 3 SSR) were linked. This resulted in 22 major linkage groups and 17 minor groups with fewer than four markers. In the Dolce Agogia map, 236 markers (93 RAPD, 133 AFLP, 6 RFLP and 4 SSR) were linked; 27 major linkage groups and three minor groups were obtained. Codominant RFLPs and SSRs, as well as few RAPDs in heteroduplex configuration, were used to establish homologies between linkage groups of both parents. The total distance covered was 2,765 cM and 2,445 cM in the Leccino and Dolce Agogia maps, respectively. The mean map distance between adjacent markers was 13.2 cM in Leccino and 11.9 cM in Dolce Agogia, respectively. Both AFLP and RAPD markers were homogeneously distributed in all of the linkage groups reported. The stearoyl-ACP desaturase gene was mapped on linkage group 4 of cv. Leccino.

Microsatellite DNA and RAPD fingerprinting, identification and genetic relationships of hybrid poplar (Populus x canadensis) cultivars.

Abstract: Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides × Populus nigra hybrids) cultivars ('Baden 431', 'Blanc du Poitou', 'Canada Blanc', 'Dorskamp 925', 'Eugenei', 'Gelrica', 'Grandis', 'Heidemij', 'I-55/56', 'I-132/56', 'I-214', 'Jacometti', 'Ostia', 'Regenerata', 'Robusta', 'Steckby' and 'Zurich 03/3'), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for 'Canada Blanc' and 'Ostia', which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars 'Baden 431', 'Heidemij', 'Robusta' and 'Steckby' are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.

Genetic Analysis of Downy Mildew Resistance Derived from Muscadinia rotundifolia

Abstract: Bulked segregant analysis was employed to identify molecular markers linked to a gene that confers downy mildew resistance to the muscadine grape. The level of resistance to downy mildew of a BC2 segregating population was estimated by visual notation after inoculation of leaves on whole plant. The broad-sense heritability of the trait was estimated at 0.88. Polymorphism revealed with RAPD (151 primers), ISSR (13 primers) and SSR (208 loci) was screened between two bulks produced by separately pooling the individual DNAs from the six most resistant and the six most susceptible plants. By analysis of variance, 1 RAPD, 4 ISSR and 8 SSR were shown to have a significant effect upon the level of resistance. Twelve of these markers were mapped on the same linkage group and cover a 45 cM long region. The identification of a QTL of resistance was confirmed by interval mapping. This QTL accounted for 73 % of the observed variation and 83% of the genetic variation. These results strongly suggest that the identified QTL corresponds to a unique major gene conditioning the muscadine grape downy mildew resistance, which we have named Rp1.Moreover, Rpv1 was shown to be tightly linked to the dominant gene confering resistance to powdery mildew, Run1.

Comparative analysis on the genetic relatedness of Sorghum bicolor accessions from Southern Africa by RAPDs, AFLPs and SSRs

Abstract: In order to get an overview on the genetic relatedness of sorghum (Sorghum bicolor) landraces and cultivars grown in low-input conditions of small-scale farming systems, 46 sorghum accessions derived from Southern Africa were evaluated on the basis of amplified fragment length polymorphism (AFLPs), random amplified polymorphic DNAs (RAPDs) and simple sequence repeats (SSRs) By this approach all sorghum accessions were uniquely fingerprinted by all marker systems Mean genetic similarity was estimated at 088 based on RAPDs, 085 using AFLPs and 031 based on SSRs In addition to this, genetic distance based on SSR data was estimated at 57 according to a stepwise mutation model (Δμ-SSR) All UPGMA-clusters showed a good fit to the similarity estimates (AFLPs: r = 092; RAPDs: r = 088; SSRs: r = 087; Δμ-SSRs: r = 085) By UPGMA-clustering two main clusters were built on all marker systems comprising landraces on the one hand and newly developed varieties on the other hand Further sub-groupings were not unequivocal Genetic diversity (H, DI) was estimated on a similar level within landraces and breeding varieties Comparing the three approaches to each other, RAPD and AFLP similarity indices were highly correlated (r = 081), while the Spearman's rank correlation coefficient between SSRs and AFLPs was r = 057 and r = 051 between RAPDs and SSRs Applying a stepwise mutation model on the SSR data resulted in an intermediate correlation coefficient between Δμ-SSRs and AFLPs (r = 066) and RAPDs (r = 067), respectively, while SSRs and Δμ-SSRs showed a lower correlation coefficient (r = 052) The highest bootstrap probabilities were found using AFLPs (56% on average) while SSR, Δμ-SSR and RAPD-based similarity estimates had low mean bootstrap probabilities (24%, 27%, 30%, respectively) The coefficient of variation (CV) of the estimated genetic similarity decreased with an increasing number of bands and was lowest using AFLPs

Development of a Specific Polymerase Chain Reaction-Based Assay for the Identification of Fusarium oxysporum f. sp. ciceris and Its Pathogenic Races 0, 1A, 5, and 6.

Abstract: Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the F. oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. F. oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of Eco RI-digested genomic DNA of a subset of F. oxysporum f. sp. ciceris and nonpathogenic F. oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F. oxysporum f. sp. ciceris races. F. oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic F. oxysporum isolates, isolates of F. oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F. oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of F. oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1A, 5, and 6.

Resistance to rust (Puccinia psidii Winter) in Eucalyptus: mode of inheritance and mapping of a major gene with RAPD markers

Abstract: Rust is one of the most-damaging eucalypt diseases in Brazil and is considered a potential threat to eucalypt plantations worldwide. To determine the mode of inheritance of resistance in the Eucalyptus grandis—Puccinia psidii pathosystem, ten full-sib families, generated from crosses between susceptible and resistant trees, were inoculated with a single-pustule isolate of the pathogen and rust severity was scored. The observed segregation ratios in segregating families suggested major gene control of rust resistance, although clearly incomplete penetrance, variable expressivity and minor genes are also involved in the global rust-resistance response. To identify markers linked to the resistance locus, screening of RAPD polymorphisms was conducted using bulked segregant analysis in a large full-sib family. A linkage group was built around the Ppr1 gene (P. psidii resistance gene 1) encompassing six RAPD markers, with a genetic window spanning 5 cM with the two most-closely linked flanking markers. Besides these two flanking markers, RAPD marker AT9/917 co-segregated with Ppr1 without a single recombinant in 994 meioses. This tightly linked marker should prove useful for marker-assisted introgression and will provide an initial lead for a positional cloning effort of this resistance allele. This is the first report of a disease resistance gene identified in Eucalyptus, and one of the few examples of the involvement of a major gene in a non-coevolved pathosystem.

A comparative study of genetic relationships among the AA-genome Oryza species using RAPD and SSR markers

Abstract: In order to estimate genetic relationships of the AA-genome Oryza species, RAPD and SSR analyses were performed with 45 accessions, including 13 cultivated varieties (eight Oryza sativa and five Oryza glaberrima) and 32 wild accessions (nine Oryza rufipogon, seven Oryza nivara, three Oryza glumaepatula, four Oryza longistaminata, six Oryza barthii, and three Oryza meridionalis). A total of 181 clear and repeatable bands were amplified from 27 selected RAPD primers, and 101 alleles were detected from 29 SSR primer pairs. The dendrogram constructed using UPGMA from a genetic-similarity matrix based on the RAPD data supported the clustering of distinct five groups with a few exceptions: O. rufipogon/O. nivara/O. meridionalis, O. barthii/O. glaberrima, O. glumaepatula, O. sativa and O. longistaminata. The dendrogram based on the SSR analysis showed a more-complicated genetic variation pattern, but the O. longistaminata and O. barthii/O. glaberrima accessions were consistently separated from all other accessions, indicating significant differentiation of the African AA-genome Oryza species. For accessions in the O. rufipogon/O. nivara/O. sativa complex, it is apparent that geographical isolation has played an important role in differentiation of the Asian AA-genome Oryza taxa. It is also demonstrated from this study that both RAPD and SSR analyses are powerful methods for detecting polymorphisms among the different AA-genome Oryza accessions. However, the RAPD analysis provides a more-informative result in terms of the overall genetic relationships at the species level compared to the SSR analysis. The SSR analysis effectively reveals diminutive variation among accessions or individuals within the same species, given approximately the same number of primers or primer-pairs used in the studies.

Molecular mapping of capsaicinoid biosynthesis genes and quantitative trait loci analysis for capsaicinoid content in Capsicum.

Abstract: Quantitative variation in the accumulation of two major capsaicinoids responsible for pungency in the fruit of chile peppers, capsaicin and dihydrocapsaicin, was analyzed in a cross between the non-pungent Capsicum annuum parent cv. Maor and a pungent Capsicum frutescens parent, accession BG 2816. In order to identify quantitative trait loci (QTLs) for capsaicinoid content, we employed the bulked segregant analysis method and screened bulked DNA from F2 individuals at the extremes of the distribution of capsaicinoid content with RAPD primers. Screening with 400 primers allowed the identification of three loci that were polymorphic between the bulks. These RAPD markers were converted to SCARs and subsequently mapped with additional RFLP markers to chromosome 7 of pepper. QTL interval analysis for individual and total capsaicinoid content identified a major QTL, termed cap, which explained 34–38% of the phenotypic variation for this trait in two growing environments. For all measurements, the allele of the pungent parent BG 2816 at cap contributed to the increased level of pungency. To determine whether known structural genes in the pathway could define a candidate for this QTL, 12 clones obtained from differentially expressed transcripts from placental tissue in pungent peppers were also mapped. None of them had a significant effect on this trait, nor did the allelic state at the locus C, the on/off switch for pungency in pepper, located on chromosome 2. The identity of cap and its effect on capsaicin content in other backgrounds will be addressed in future studies.

Escherichia coli Strains from Pregnant Women and Neonates: Intraspecies Genetic Distribution and Prevalence of Virulence Factors

Abstract: To determine the extent to which the vagina, endocervix, and amniotic fluid screen the Escherichia coli strains responsible for neonatal infections, we studied the genetic relationships among 105 E. coli strains isolated from all of the ecosystems involved in this infectious process. Twenty-four strains were isolated from the intestinal flora, and 25 strains were isolated from the vaginas of pregnant women. Twenty-seven strains were isolated from the amniotic fluid, blood, and cerebrospinal fluid (CSF) of infected neonates. The intraspecies genetic characteristics of all of the isolates were determined by random amplified polymorphic DNA (RAPD) analysis, PCR ECOR (E. coli reference) grouping, and PCR virulence genotyping. A correlation was found between the intraspecies distributions of the strains in the A, B1, B2, and D ECOR groups and in the two major RAPD groups (I and II). Nevertheless, the distribution of the E. coli strains in the RAPD groups according to their anatomical origins was more significant than their distribution in the ECOR groups. This may be explained by the existence of an E. coli subpopulation, defined by the RAPD I group, within the ECOR B2 group. This RAPD I group presents a major risk for neonates: 75% of the strains isolated from patients with meningitis and 100% of the strains isolated from patients with bacteremia were in this group. The vagina and the amniotic fluid are two barriers that favor colonization by highly infectious strains. Indeed, only 17% of fecal strains belonged to the RAPD I group, whereas 52% of vaginal strains and 67% of amniotic fluid strains belonged to this subpopulation. The ibeA and iucC genes were significantly associated with CSF strains, whereas the hly and sfa/foc genes were more frequent in blood strains. These findings could serve as a basis for developing tools to recognize vaginal strains, which present a high risk for neonates, for use in prophylaxis programs.

Comparison of AFLPs, RAPD Markers, and Isozymes for Diversity Assessment of Garlic and Detection of Putative Duplicates in Germplasm Collections

Abstract: Garlic (Allium sativum L.) is an asexually propagated crop that displays much morphological diversity. Studies which have assessed garlic diversity with isozymes and randomly amplified polymorphic DNA (RAPD) markers generally agreed with the morphological observations but sometimes failed to discriminate clones. To discriminate among closely related garlic clones in more detail, we introduced amplified fragment-length polymorphism (AFLPs) to evaluate the genetic diversity and phenetic relatedness of 45 garlic clones and three A. longicuspis clones and we compared AFLP results with RAPD markers and isozymes. Three AFLP primer combinations generated a total of 183 polymorphic fragments. Although similarities between the clusters were low (≥0.30), some clones within the clusters were very similar (>0.95) with AFLP analysis. Sixteen clones represented only six different banding patterns, within which they shared 100% polymorphic AFLPs and RAPD markers, and likely are duplicates. In agreement with the results of other investigators, A. longicuspis and A. sativum clones were clustered together with no clear separation, suggesting these species are not genetically or specifically distinct. The topology of AFLP, RAPD, and isozyme dendrograms were similar, but RAPD and isozyme dendrograms reflected less and much less polymorphism, respectively. Comparison of unweighted pair group method with arithmetic averaging (UPGMA) dendrograms of AFLP, RAPD, and isozyme cluster analyses using the Mantel test indicated a correlation of 0.96, 0.55, and 0.57 between AFLP and RAPD, AFLP and isozyme, and RAPD and isozyme, respectively. Polymorphic AFLPs are abundant in garlic and demonstrated genetic diversity among closely related clones which could not be differentiated with RAPD markers and isozymes. Therefore, AFLP is an additional tool for fingerprinting and detailed assessment of genetic relationships in garlic. longicuspis clones were discriminated morphologically from A. sativum clones by generally having exerted and purple anthers, higher flowering rate and seed production, and smaller bulbils in inflorescence (Etoh and Simon, 2002). The species have the same karyotype and A. sativum and A. longicuspis clones have been crossed successfully (Etoh, 1984; Pooler and Simon, 1994). Garlic has been propagated by vegetative means for centuries and the presence of many closely related or duplicated garlic clones is likely in germplasm collections. Large numbers of molecular markers are needed to discriminate closely related clones or to identify duplicates. One particularly useful tool to achieve this goal is amplified fragment-length polymorphism (AFLP), which could produce high multiplex informative mark- ers in a single reaction and, therefore, could generate sufficient markers to assess genetic diversity among garlic clones. AFLP is a PCR-based DNA fingerprinting technique (Vos et al., 1995) and like randomly amplified polymorphic DNA (RAPD), it generates primarily dominant markers. Neither AFLP nor RAPD require any probe or sequence information (Barker et al., 1999) and they can be applied to any species after some minor modifications. Isozymes, RAPD, and AFLP have been used extensively for determining genetic diversity and relatedness

Molecular and morphological characterisation of the Heterodera avenae species complex (Tylenchida: Heteroderidae)

Abstract: Species of the Heterodera avenae complex, including populations of H. arenaria, H. aucklandica, H. australis, H. avenae, H. filipjevi, H. mani, H. pratensis and H. ustinovi, obtained from different regions of the world were analysed with PCR-RFLP and sequencing of the ITS-rDNA, RAPD and light microscopy. Phylogenetic relationships between species and populations of the H. avenae complex as inferred from analyses of 70 sequences of the ITS region and of 237 RAPD markers revealed that the cereal cyst nematode H. avenae is a paraphyletic taxon. The taxonomic status of the Australian cereal cyst nematode H. australis based on sequences of the ITS-rDNA and RAPD data is confirmed. Morphometrical and ITS-rDNA sequence analyses revealed that the Chinese cereal cyst nematode is different from other H. avenae populations infecting cereals and is related to H. pratensis. Bidera riparia Kazachenko, 1993 is transferred to the genus Heterodera as H. riparia (Kazachenko, 1993) comb. n. As a consequence, H. riparia Subbotin, Sturhan, Waeyenberge & Moens, 1997 becomes a junior secondary homonym and is renamed as H. ripae nom. nov. Morphological, morphometrical characters and RFLP profiles for identification of the nine species presently placed in the H. avenae species complex are given.

The use of molecular markers for germplasm management in a French olive collection.

Abstract: With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rhone Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers.

Abstract: Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism Out of 124 polymerase chain reaction fragments that were scored, 120 (968%) were polymorphic All the plants could be discriminated and constitute a very heterogeneous group Five unidentified almond plants found in the region of Foz Coa (north Portugal) and wild almond (P webbii) from Italy and Spain were also included Four main groups of plants could be distinguished: P dulcis cultivars; one Foz Coa plant; P webbii; and P persica (outgroup) The segregating Foz Coa plant may represent a feral individual or a hybrid between P dulcis and P webbii

Analysis of Sweet Cherry (Prunus avium L.) Cultivars Using SSR and AFLP Markers

Abstract: Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evalu- ate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite contain- ing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of 'Bing∑ fruit collected from different grocery stores in the United States to that of a standard 'Bing∑ cultivar. Results indicated that, with one exception, all 'Bing∑ samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study. The capability to distinguish among cherry (Prunus avium) culti- vars in breeding and cultivation is extremely important for scientific as well as for economic reasons. There is a demand for a rapid and reliable method of cultivar identification for Plant Breeder∑ s Rights (PBR) registration and protection. Classical methods of identifica- tion and characterization of cultivars in fruits have relied mostly upon a large set of phenotypic data that is often difficult to assess, may vary with environment and production practices, and can be time consuming to collect when surveying large populations that may be growing in different locations. Molecular markers based on DNA are stable, detectable in all tissues and independent of environmental or seasonal conditions. They can be used for cultivar identification, diversity analysis, assessment of parentage, patent issues and quality control of rootstock-seed lots. The ability to distinguish cherry cultivars would be greatly enhanced by the use of molecular markers. Molecular marker development and use within Prunus species has been most active in peach (P. persica) due to its relatively short juvenile period and commercial importance (Dirlewanger and Bodo, 1994; Sosinski et al., 1998). A variety of molecular techniques have been developed for measuring genetic variabil- ity. Of the possible alternatives, isozymes have been shown to be insufficiently variable in cherry due to low polymorphism of the species ( Tobutt and Boskovic, 1996). Randomly amplified poly- morphic DNA (RAPD) markers have also been assayed in fruit

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting.

Abstract: Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59-0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66-0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.

Use of an Open-Reading Frame–Specific Campylobacter jejuni DNA Microarray as a New Genotyping Tool for Studying Epidemiologically Related Isolates

Abstract: Findings from use of an open-reading frame-specific Campylobacter jejuni DNA microarray to investigate genetic diversity among clinical isolates associated with 5 independent clusters of infection were compared with data from random amplified polymeric DNA (RAPD) and Penner serotyping analyses. The DNA microarray provides a highly specific epidemiological typing tool for analysis of C. jejuni isolates and reveals both divergent and highly conserved gene classes among isolates.

Characterization of sourdough lactic acid bacteria based on genotypic and cell‐wall protein analyses

Abstract: Aims: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. Methods and Results: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. Conclusions: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. Significance and Impact of the Study: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.